1. Field of the Invention
This invention relates to methods and apparatus for performing microanalytic and microsynthetic analyses and procedures. In particular, the invention relates to microminiaturization of genetic, biochemical and bioanalytic processes. Specifically, the present invention provides devices and methods for the performance of miniaturized biochemical assays. These assays may be performed for a variety of purposes, including but not limited to screening of drug candidate compounds, life sciences research, and clinical and molecular diagnostics. Methods for performing any of a wide variety of such microanalytical or microsynthetic processes using the microsystems apparatus of the invention are also provided.
2. Background of the Related Art
Recent developments in a variety of investigational and research fields have created a need for improved methods and apparatus for performing analytical, particularly bioanalytical assays at microscale (i.e., in volumes of less than 100 xcexcL). In the field of pharmaceuticals, for example, an increasing number of potential drug candidates require assessment of their biological function. As an example, the field of combinatorial chemistry combines various structural sub-units with differing chemical affinities or configurations into molecules; in theory, a new molecule having potentially unique biochemical properties can be created for each permutation of the sub-units. In this way, large libraries of compounds may be synthesized from relatively small numbers of constituents, each such compound being a potential drug lead compound of usually unknown biological activity and potency.
More traditional approaches to compound library development are also yielding growing numbers of candidates, including the use of naturally-derived compounds extracted from plants, fungi, and bacteria. In part, this is due to a growing understanding of the function of these compounds, including how they affect the metabolic pathways of the organisms which synthesize and use them; the increasing refinement in identifying and understanding compounds based on small structural and compositional differences; and improved methods for extracting and purifying these compounds.
Increased numbers of potential targets for these drug candidates are also being identified. Recent advances in biology, most notably the human genome project, have discovered many molecules whose biochemical activity is implicated in various disease states. Although these novel targets can provide exquisitely precise and specific indicia of how biological processes underlying disease can be effectively controlled and manipulated, drugs must be identified, usually by screening processes, to find compounds that can enhance, diminish, or otherwise alter these targets"" ability to affect the metabolic pathways associated with disease.
The function of drug candidates, targets, and the effect of the candidates on targets is assessed in the early stages of pharmaceutical development through a process of screening that typically includes: binding of a drug candidate to a portion or domain of the target molecule; immunoassays that bind to drug candidate target domains correlated with drug efficacy; enzymatic assays, in which the inhibition of an enzymatic activity of the target by the drug candidate can be used as a sign of efficacy; protein/protein binding; and protein/DNA(RNA) binding. Additional assays involve the use of living cells and include gene expression, in which levels of transcription in response to a drug candidate are monitored, and functional assays designed to investigate both macroscopic effects, such as cell viability, as well as biochemical effects and products produced in and by the cells as a result of treatment with the drug lead compound. (Wallace and Goldman, 1997, xe2x80x9cBioassay Design and Implementationxe2x80x9d, in High-Throughput Screening: The Discovery of Bioactive Substances, J. P. Devlin, ed., Marcel Dekker, Inc.: New York, pp. 279-305).
In initial screening of compounds against targets, the number of possible screens is roughly the number of candidates multiplied by the number of targets. As a result of the growth in both the number of candidates and the number of targets, the number of assays that must be performed is growing rapidly. In addition to the increasing the number of assays to be performed, it is desirable to reduce the time required to perform the assays in order to obtain results of such screenings in a timely and useful fashion. Finally, xe2x80x9cmultiplexingxe2x80x9d technology that allows the performance of multiple assays on one sample within a single reaction wellxe2x80x94for example, by using readily-distinguishable signals, such as fluorescent moieties with different characteristic wavelengthsxe2x80x94can be used to increase throughput.
In addition to drug screening assays, biological research has uncovered a vast reservoir of genetic information and diversity having little if any correlation with the function of the gene products encoded by the deciphered DNA. On the one hand, the identification of the nucleotide sequence of the human genome, coupled with bioinformatics analysis of these sequences, has identified a larger number of protein coding sequences (termed xe2x80x9copen reading framesxe2x80x9d) that can and probably do encode functional proteins. However, since these sequences have been uncovered by simply xe2x80x9creadingxe2x80x9d a sequence without any information (such as the correlation of a genetic locus with a mutation associated with a disease), the function of the gene products of such a locus must be determined in order to fully understand and identify what protein target is encoded thereby and what utility drug candidates directed to such a target might have. On the other hand, human genome sequencing efforts have also identified genetic mutations (such as single nucleotide polymorphisms, or xe2x80x9cSNPsxe2x80x9d) that may or may not be associated with human disease. In either instance, the products of this human genetic information must be assayed to determine the activity of the genes, both xe2x80x9cwild-typexe2x80x9d and mutant, encoded at each new genetic locus. Progress in life sciences research requires researchers to perform large numbers of assays as they investigate the structure and function of proteins coded by the growing number of identified genes in the human genome. Many of the same assays and assay formats used in drug screening may be used in other life sciences research.
Large numbers of assays must also be performed in the field of molecular diagnostics, in which individuals can now be assayed for genetic mutation associated with a disease state or the propensity to develop a disease state. For example, any particular disease or propensity for disease may be associated with several different mutations in more than one gene that can determine disease susceptibility or severity. In the monitoring of a disease state, a disease may have a xe2x80x9cfingerprintxe2x80x9d consisting of certain genes the expression level of which can be used diagnostically to predict the severity of the disease. Monitoring expression levels of these genes can provide an indication of the response (or lack of response) to different treatment modalities.
For these and other applications in drug discovery, life sciences research, and molecular and clinical diagnostics there exists a need for systems and assay methods that can perform very many assays in a highly-parallel fashion at low cost. The primary approach has been and will continue to be to miniaturize existing assays in order to decrease compound and reagent costs (that scale with the volume required for performing the assay). Miniaturization has been accompanied by the development of more sensitive detection schemes, including both better detectors for conventional signals (e.g., calorimetric absorption, fluorescence, and chemiluminescence) as well as new chemistries or assay formats (e.g., imaging, optical scanning, and confocal microscopy).
Miniaturization can also confer performance advantages. At short length scales, diffusionally-limited mixing is rapid and can be exploited to create sensitive assays (Brody et al., 1996, Biophyscal J. 71: 3430-3431). Because fluid flow in miniaturized pressure-driven systems is laminar, rather than turbulent, processes such as washing and fluid replacement are well-controlled. Microfabricated systems also enable assays that rely on a large surface area to volume ratio such as those that require binding to a surface and a variety of chromatographic approaches
The development of fluid-handling and processing for miniaturized assays has primarily involved scaling down of conventional methods. The vast majority of initial drug screens have been performed in 96-well microtiter plates with operating volumes of less than 0.5 mL. The wells of these plates serve as xe2x80x9ctest tubesxe2x80x9d for reactions as well as optical cuvettes for detection. Fluids are typically delivered to these plates using automated pipetting stations or external tubing and pumps; automation is also required for handling of plates and delivery to sub-systems such as plate washers (used in solid phase assays, for example).
Miniaturization has led to the creation of 384-well and 1536-well microtiter plates for total reaction volumes of between 0.015 and 0.1 mL. However, a number of problems arise when miniaturizing standard plate technology. First, because the total volumes are smaller and the plates are open to the environment, evaporation of fluid during the course of an assay can compromise results. Another drawback of open plates is the existence of a fluid meniscus in the well. Meniscuses of varying configurations (due, for example to imperfections in the plate or differences in contact angle and surface tension) can distort the optical signals used to interrogate the samples. As the strength of the optical signals decreases with decreasing assay volume, correction for background distortions becomes more difficult. Finally, optical scanning systems for high-density plates are often complex and expensive. Methods that minimize evaporation, provide a more uniform optical pathway, and provide simpler detection schemes are desirable.
Highly accurate pipetting technologies have been developed to deliver fluids in precisely metered quantities to these plates. Most of these fluid-delivery methods for low volumes (below a few microliters) rely on expensive piezoelectric pipetting heads that are complex and difficult to combine or xe2x80x9cgangxe2x80x9d into large numbers of independent pipettors so that many wells may be addressed independently. As a result, fluid delivery is either completely or partially serial (i.e., a single micropipettor, or a small number of parallel delivery systems used repeatedly to address the entire plate). Serial pipetting defeats the aim of parallelism by increasing the amount of time required to address the plate. Methods that reduce the number and precision of fluid transfer steps are therefore needed.
Fluid processing in microtiter plates is also difficult. The small dimensions of the wells, while enhancing diffusional mixing, suppress turbulence and make difficult mixing on length scales between a few tens of microns and a few millimeters. For similar reasons, washing, an important step in many assays can be problematic. Methods that reduce both the number of manipulations of fluids on the plate as well as manipulations of the plate itself (such as passing the plate to and from washing stations) can reduce cost while improving assay quality through suppression of contamination, carry-over, and fluid loss.
Thus, there is a need in the art for improved micromanipulation apparatus and methods for performing bioanalytic assays more rapidly and economically using less biological sample material. Relevant to this need in the art, some of the present inventors have developed a microsystem platform and a micromanipulation device to manipulate said platform by rotation, thereby utilizing the centripetal forces resulting from rotation of the platform to motivate fluid movement through microchannels embedded in the microplatform, as disclosed in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Ser. No. 08/761,063, filed Dec. 5, 1996; U.S. Ser. No. 08/768,990, filed Dec. 18, 1996; U.S. Ser. No. 08/910,726, filed Aug. 12, 1997; U.S. Ser. No. 08/995,056, filed Dec. 19, 1997; and U.S. Pat. No. 09/315,114, filed May 19, 1999, the disclosures of each of which are explicitly incorporated by reference herein.
This invention provides microsystems platforms as disclosed in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Ser. No. 08/761,063, filed Dec. 5, 1996; U.S. Ser. No. 08/768,990, filed Dec. 18, 1996; U.S. Ser. No. 08/910,726, filed Aug. 12, 1997; U.S. Ser. No. 08/995,056, filed Dec. 19, 1997; and U.S. Ser. No. 09/315,114,filed May 19, 1999, the disclosures of each of which are explicitly incorporated by reference herein.
The invention provides apparatus and methods for performing microscale processes on a microplatform, whereby fluid is moved on the platform in defined channels motivated by centripetal force arising from rotation of the platform. The first element of the apparatus of the invention is a microplatform that is a rotatable structure, most preferably a disk, the disk comprising fluid (sample) inlet ports, fluidic microchannels, reagent reservoirs, collection chambers, detection chambers and sample outlet ports, generically termed xe2x80x9cmicrofluidic structures.xe2x80x9d The disk is rotated at speeds from about 1 to about 30,000 rpm for generating centripetal acceleration that enables fluid movement through the microfluidic structures of the platform. The disks of the invention also preferably comprise air outlet ports and air displacement channels. The air outlet ports and in particular the air displacement ports provide a means for fluids to displace air, thus ensuring uninhibited movement of fluids on the disk. Specific sites on the disk also preferably comprise elements that allow fluids to be analyzed, as well as detectors for each of these effectors.
The discs of this invention have several advantages over those that exist in the centrifugal analyzer art. Foremost is the fact that flow is laminar due to the small dimensions of the fluid channels; this allows for better control of processes such as mixing and washing. Secondly, the small dimensions conferred by microfabrication enable the use of xe2x80x9cpassivexe2x80x9d valving, dependent upon capillary forces, over much wider range of rotational velocities and with greater reliability than in more macroscopic systems. To this are added the already described advantages of miniaturization.
The second element of the invention is a micromanipulation device that is a disk player/reader device that controls the function of the disk. This device comprises mechanisms and motors that enable the disk to be loaded and rotated. In addition, the device provides means for a user to operate the microsystems in the disk and access and analyze data, preferably using a keypad and computer display. The micromanipulation device also advantageous provides means for actuation of on-disc elements, such active valves; the application and control of heat to the disc for purposes of chemical or biological incubation; and means for adding fluids to and removing fluids from the discs. The micromanipulation devices of this invention are more particularly described in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Ser. No. 08/761,063, filed Dec. 5, 1996; U.S. Ser. No. 08/768,990, filed Dec. 18, 1996; U.S. Ser. No. 08/910,726, filed Aug. 12, 1997; U.S. Ser. No. 08/995,056, filed Dec. 19, 1997; and U.S. Ser. No. 09/315,114, filed May 19, 1999, the disclosures of each of which are explicitly incorporated by reference herein.
The invention specifically provides microsystems platforms comprising microfluidics components contained in one or a multiplicity of platform layers that are fluidly connected to permit transfer, mixing and assay performance on the sealed surface of the platform. The platforms preferably comprise reagent reservoirs containing a sufficient volume, preferably from about 1 nL to about 1 mL, of a reagent solution for a multiplicity of individual assays. The reagent reservoirs are fluidly connected by microchannels to one or more preferably a multiplicity of collection, and more preferably detection, chambers, and the microfluidics components arranged so that a specific volume of the reagent solution is delivered to each collection chamber. More preferably, said reagent reservoirs are fluidly connected to mixing structures, most preferably a mixing microchannel that is also fluidly connected to a sample reservoir, so that one or a plurality of reagents are mixed with sample and the resulting mixture delivered into the detection chamber. In preferred embodiments, the platform comprises a multiplicity of sample reservoirs and mixing structures fluidly connected with a multiplicity of detection chambers.
In the use of the platforms of the invention, fluids (including samples and reagents) are added to the platform when the platform is at rest. Thereafter, rotation of the platform on a simple motor motivates fluid movement through microchannels for various processing steps. In preferred embodiments, the platforms of the invention permit the use of a detector, most preferably an optical detector, for detecting the products of the assay, whereby the collection chambers comprise optical cuvettes, preferably at the outer edge of the platform, most preferably wherein the platform is scanned past a fixed detector through the action of the rotary motor. Because the platforms of the invention are most preferably constructed using microfabrication techniques as described more fully below, the volumes of fluids used may be made arbitrarily small as long as the detectors used have sufficient sensitivity.
The present invention solves problems in the current art through the use of a microfluidic disc in which centripetal acceleration is used to move fluids. It is an advantage of the microfluidics platforms of the present invention that the fluid-containing components are constructed to contain a small volume, thus reducing reagent costs, reaction times and the amount of biological material required to perform an assay. It is also an advantage that the fluid-containing components are sealed, thus eliminating experimental error due to differential evaporation of different fluids and the resulting changes in reagent concentration. Because the microfluidic devices of the invention are completely enclosed, both evaporation and optical distortion are reduced to negligible levels. The platforms of the invention also advantageously permit xe2x80x9cpassivexe2x80x9d mixing and valving, i.e., mixing and valving are performed as a consequence of the structural arrangements of the components on the platforms (such as shape, length, position on the platform surface relative to the axis of rotation, and surface properties of the interior surfaces of the components, such as wettability as discussed below), and the dynamics of platform rotation (speed, acceleration, direction and change-of-direction), and permit control of assay timing and reagent delivery.
In alternative embodiments of the platforms of the invention, metering structures as disclosed in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000 and incorporated by reference herein, are used to distribute aliquots of reagent to each of a multiplicity of mixing structures, each mixing structure being fluidly connected to one of a multiplicity of sample reservoirs, thereby permitting parallel processing and mixing of the samples with a common reagent. This reduces the need for automated reagent distribution mechanisms, reduces the amount of time required for reagent dispensing (that can be performed in parallel with distribution of reagent to a multiplicity of reaction chambers), and permits delivery of small (nL-to-xcexcL) volumes without using externally-applied electromotive means.
The assembly of a multiplicity of collection chambers on the platforms of the invention also permits simplified detectors to be used, whereby each individual collection/detection chamber can be scanned using mechanisms well-developed in the art for use with, for example, CD-ROM technology. Finally, the platforms of the invention are advantageously provided with sample and reagent entry ports for filling with samples and reagents, respectively, that can be adapted to liquid delivery means known in the art (such as micropipettors).
The platforms of the invention reduce the demands on automation in at least three ways. First, the need for precise metering of delivered fluids is relaxed through the use of on-disc metering structures, as described more fully in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Ser. No. 08/761,063, filed Dec. 5, 1996; U.S. Ser. No. 08/768,990, filed Dec. 18, 1996; U.S. Ser. No. 08/910,726, filed Aug. 12, 1997; U.S. Ser. No. 08/995,056, filed Dec. 19, 1997; and U.S. Ser. No. 09/315,114, filed May 19, 1999, the disclosures of each of which are explicitly incorporated by reference herein. By loading imprecise volumes, slightly in excess of those needed for the assay, and allowing the rotation of the disc and use of appropriate microfluidic structures to meter the fluids, much simpler (and less expensive) fluid delivery technology may be employed than is the conventionally required for high-density microtitre plate assays.
Second, the total number of fluid xe2x80x9cdeliveryxe2x80x9d events on the microfluidic platform is reduced relative to microtiter plates. By using microfluidic structures that sub-divide and aliquot common reagents (such as reagent solutions, buffers, and enzyme substrates) used in all assays performed on the platform, the number of manual or automated pipetting steps are reduced by at least half (depending on the complexity of the assay). A reduction in fluid transfers to the device can reduce total assay time. Examples of these structures have been disclosed in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and incorporated by reference herein.
Finally, the invention also provides on-platform means for mixing reagents with sample and washing the resulting reaction products, removing the need for transferring the assay collection chamber(s) to a separate xe2x80x9cwashxe2x80x9d station. This also reduces manipulation of the assay device as well as providing controlled and integrated fluid processing.
Certain preferred embodiments of the apparatus of the invention are described in greater detail in the following sections of this application and in the drawings.